Anti-pd-1 antibody and use thereof

ABSTRACT

This invention provides antibodies or functional fragments thereof that bind to PD-1 with high affinity. The invention provides nucleic acid molecules encoding the antibodies or the fragments thereof according to the present invention, expression vectors and host cells for expressing the antibodies or the functional fragments thereof according to the present invention, as well as methods for producing the antibodies or the functional fragments thereof according to the present invention. The present invention also provides immunoconjugates and pharmaceutical compositions comprising the antibodies or the functional fragments thereof according to the present invention. The present invention additionally provides methods for treating a plurality of diseases (comprising cancers, infectious diseases and inflammatory diseases) by using the antibodies or the functional fragments thereof disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 16/045,363, with a filingdate of Jul. 25, 2018, which is a continuation of Ser. No. 14/392,360,with a filing date of Jun. 7, 2016, now patent Ser. No. 10/066,013,issued on Sep. 4, 2018, which is a National Stage Entry ofPCT/CN2014/072574, with a filing date of Feb. 26, 2014, which claimspriority to Chinese Patent Application No. 201310258289.2, with a filingdate of Jun. 26, 2013, all of which this application incorporates byreference herein.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in file098903-1097645-000110US_SequenceListing.txt created on Jul. 24, 2018,32,644 bytes, machine format IBM-PC, MS-Windows operating system, inaccordance with 37 C.F.R. §§ 1.821- to 1.825, is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION Technical Field

The present invention belongs to the field of biomedicine and relates toantibodies or functional fragments thereof that bind specifically toPD-1 with high affinity. The invention provides nucleic acid moleculesencoding the antibodies or the fragments thereof according to thepresent invention, expression vectors and host cells for expressing theantibodies or the functional fragments thereof according to the presentinvention, as well as methods for producing the antibodies or thefunctional fragments thereof according to the present invention. Thepresent invention also provides immunoconjugates and pharmaceuticalcompositions comprising the antibodies or the functional fragmentsthereof according to the present invention. The present inventionadditionally provides methods for treating a plurality of diseases(comprising cancers, infectious diseases and inflammatory diseases) byusing the antibodies or the functional fragments thereof disclosedherein.

Description of Related Art

The programmed cell death 1 protein, PD-1, is a member of CD28 familyand an immunosuppressive receptor expressed on the surfaces of theactivated T cells and B cells (Yao, Zhu et al., Advances in targetingcell surface signaling molecules for immune modulation. Nat Rev DrugDiscov, 2013, 12(2): 130-146). This receptor can bind to its ligandsPD-L1 and PD-L2 to effectively reduce the immune response involving Tcells. Tumor cells can escape the immune surveillance inside the bodyvia high expression of PD-L1 (Okazaki and Honjo, PD-1 and PD-1 ligands:from discovery to clinical application. International Immunology, 2007,19(7): 813-824 2007). The interaction between PD1 and PD-L1 can beblocked to significantly improve the tumor-killing activity of theCD8+cytotoxic T cells.

PD-1 is primarily expressed on the surface of CD4+ T cells, CD8+ Tcells, NKT cells, B cells, and the activated monocytes. The expressionof PD-1 is primarily induced by the signals of T cell receptor (TCR) orB cell receptor (BCR). TNF can enhance the expression of PD-1 on thesurfaces of these cells (Francisco, Sage et al., The PD-1 pathway intolerance and autoimmunity. Immunol Rev, 2010, 236: 219-242). Human PD-1is encoded by the gene Pdcd1, which is located on 2q37.3 and is 9.6 kb.It comprises five exons and four introns and its upstream comprises apromoter of 663 bp. The molecular structure of PD-1 comprisesextracellular region, trans-membrane region and intracellular region.The amino acid sequences in the extracellular region has 24% homologywith CTLA-4 and 28% homology with CD28. Its gene has primarily sevensingle-nucleotide polymorphic sites. The extracellular region comprisesone structural domain of immuno-globulin variable IgV. The intracellularregion comprises two signal transduction motifs based on tyrosine—ITIM(immunoreceptor tyrosine-based inhibitory motif) and ITSM(immunoreceptor tyrosine-based conversion motif). Once T cells areactivated, PD-1 will associate with tyrosine phosphatase SHP2 primarilyvia the ITSM motif to cause the de-phosphorylation of the effectormolecules including CD3ζ, PKCθ and ZAP70, etc.

There are two PD-1 ligands: PD-L1 and PD-L2. PD-L1 is also referred toas B7H1 or CD274 and PD-L2 is referred to as B7DC or CD273. The PD-Lgene is located on the locus of 9p24.2 of human chromosome with a sizeof 42 kb. These ligands have 21-27% homology in amino acid sequence andstructural similarity with B7-1, B7-2 and ICOSL. The PD-1 ligands allcomprise one structural domain of immuno-globulin-like variable region,one constant-region-like structural domain, one trans-membrane region,and one short cytoplast tail. The cytoplast tail of PD-L1 is moreconservative than that of PD-L2. PD-L1 and PD-L2 are expressed ondifferent cell populations (Shimauchi, Kabashima et al., Augmentedexpression of programmed death-1 in both neoplastic and non-neoplasticCD4+ T cells in adult T cell leukemia/lymphoma. Int J Cancer, 2007,121(12): 2585-2590). These cells include non-hematopoietic tissue and avariety of tumor types. PD-L1 is mainly expressed on T cells, B cells,dendritic cells, macrophages, mesenchymal stem cells and mast cellsderived from bone marrow. PD-L1 is also expressed on the cells notderived from bone marrow, such as vascular endothelial cells, epithelialcells, skeletal muscle cells, hepatocytes, renal tubular epithelialcells, islet cells, brain astrocytes, and various types of non-lymphoidtumors such as melanoma, liver cancer, stomach cancer, renal cellcarcinoma, as well as expressed on the cells at the immunologicallyprivileged sites such as placenta, eyes. It has been suggested thatPD-L1 can be extensive to some degree in regulating the auto-reactive Tcells, B cells and immune tolerance and can play a role in response ofperipheral tissue T cells and B cells. Nevertheless, the PD-L2 has verylimited expressed region and exists only in macrophages and dendriticcells. PD-L1 is believed to play a role mainly in the immunepresentation.

PD-1 and PD-L1 interact with each other to regulate and control theactivation of T cells, which has been validated by much in tumors andviral infections. PD-L1 is expressed on the surfaces of various tumorcells which include lung cancer, liver cancer, ovarian cancer, cervicalcancer, skin cancer, bladder cancer, colon cancer, breast cancer,glioma, renal carcinoma, stomach cancer, esophageal cancer, oralsquamous cell cancer, and head/neck cancer. There are large amount ofCD8+ T cells expressing PD-L1 found around these cancers. The clinicalstatistics reveal that the high level of expression of PD-L1 on cancercells is related to the poor prognosis of cancer patients (Okazaki andHonjo 2007. Supra.).

Many chronic and acute viruses also escape the body's immunesurveillance via the signals of PD-1 and PD-L1. For example, theexpression level of PD-1 in HIV-infected patients is closely related tothe degree of depletion of T cells and can be used as one of the markersof AIDS progression (Trabattoni, Saresella et al., B7-H1 is up-regulatedin HIV infection and is a novel surrogate marker of disease progression.Blood, 2003, 101(7): 2514-2520). It is the same case for the patientsdiagnosed with chronic hepatitis B (Evans, Riva et al., Programmed death1 expression during antiviral treatment of chronic hepatitis B: Impactof hepatitis B e-antigen seroconversion. Hepatology, 2008, 48(3):759-769). The animal tests revealed that the mice whose PD-1 gene hasbeen knocked out can control virus infection better than the normalmice; and hepatitis can be induced if the HBV-specific T cells aretransferred into the HBV transgenic animals.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an anti-PD-1 antibody or a functionalfragment thereof which can bind to the programmed cell death 1 (PD-1).

In one aspect, the antibody or the functional fragment thereof comprisesa heavy chain CDR selected from the amino acid sequence SEQ ID NO: 1, 2,3, 7, 8, 9, 13, 14, 15 or any variant of said sequence, and/or a lightchain CDR selected from the amino acid sequence SEQ ID NO: 4, 5, 6, 10,11, 12, 16, 17, 18 or any variant of said sequence.

In some preferred embodiments, the antibody or the functional fragmentthereof comprises a) heavy chain CDR1, CDR2 and CDR3 sequences selectedfrom any of the following groups of various amino acid sequences ortheir variants:

HCDR1 HCDR2 HCDR3 A SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 B SEQ ID NO:7 SEQ ID NO: 8 SEQ ID NO: 9 C SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15

and/or light chain CDR1, CDR2 and CDR3 sequences selected from any ofthe following groups of various amino acid sequences and their variants:

LCDR1 LCDR2 LCDR3 A SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 B SEQ ID NO:10 SEQ ID NO: 11 SEQ ID NO: 12 C SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO:18

In some preferred embodiments, the amino acid sequences of the heavychain CDR1, CDR2 and CDR3 as well as the light chain CDR1, CDR2 and CDR3of the antibody or the functional fragment thereof according to thepresent invention are selected from any of the following groups ofvarious amino acid sequences or their variants:

HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 A SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 6 B SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID NO: 7 NO: 8 NO: 9 NO: 10 NO: 11 NO: 12 C SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID NO: 13 NO: 14 NO: 15 NO: 16 NO: 17 NO: 18

In some embodiments, the antibody or the functional fragment thereofaccording to the present invention comprises a) a variable region of theheavy chain selected from the group consisting of amino acid sequencesSEQ ID NOs: 19, 21, 23, and any variant of SEQ ID NO: 19, 21, or 23.and/or b) a variable region(s) of the light chain selected from theamino acid sequence SEQ ID NO: 20, 22, 24 or any variant of saidsequence.

In one preferred embodiment, said variable region of the heavy chain isSEQ ID NO: 19 or any variant thereof and said light chain is SEQ ID NO:20 or any variant thereof.

In another preferred embodiment, said variable region of the heavy chainis SEQ ID NO: 21 or its variant and said variable region of the lightchain is SEQ ID NO: 22 or any variant thereof.

In another more preferred embodiment, said variable region of the heavychain is SEQ ID NO: 23 or any variant thereof and said variable regionof the light chain is SEQ ID NO: 24 or any variant thereof.

The antibody or the functional fragment thereof according to the presentinvention can be a chimeric antibody, a humanized antibody or a fullyhuman antibody.

The antibody or the functional fragment thereof according to the presentinvention can be humanized. Methods of preparing humanized antibody aregenerally known by the skilled in the art. For example, the CDR sequenceaccording to the present invention can be transferred into the variableregion of a human antibody to prepare the humanized anti-PD-1 antibodyof the present invention. Said humanized antibody will not produce antiantibody response (AAR) and human anti-mouse antibody (HAMA) response,and will not be removed quickly due to neutralization by anti antibodyand will play a role of immunological effect such as ADCC and CDCeffects.

In some preferred embodiments, the humanized PD-1 antibody or thefunctional fragment thereof according to the present invention comprisesa) a variable region of the heavy chain selected from the groupconsisting of amino acid sequences SEQ ID NO: 33, 35, 36 and any variantof SEQ ID NO: 33, 35, or 36, and/or b) a variable region of the lightchain selected from SEQ ID NO: 34, 37 and any variant of SEQ ID NO: 34or 37.

In one preferred embodiment of the humanized antibody or the functionalfragment thereof according to the present invention, said variableregion of the heavy chain is SEQ ID NO: 33 or its variant and saidvariable region of the light chain is SEQ ID NO: 34 or its variant.

In another preferred embodiment of the humanized antibody or thefunctional fragment thereof according to the present invention, saidvariable region of the heavy chain is SEQ ID NO: 35 or its variant andsaid variable regions of the light chain is SEQ ID NO: 34 or itsvariant.

In another preferred embodiment of the humanized antibody or thefunctional fragment thereof according to the present invention, saidvariable region of the heavy chain is SEQ ID NO: 36 or its variant andsaid variable region of the light chain is SEQ ID NO: 34 or its variant.

In another more preferred embodiment of the humanized antibody or thefunctional fragments thereof according to the present invention, saidvariable region of the heavy chain is SEQ ID NO: 35 or its variant andsaid variable region of the light chain is SEQ ID NO: 37 or its variant.

The present invention also provides a separate nucleic acid moleculeencoding the antibody or the functional fragment thereof according tothe present invention. In one preferred embodiment, said nucleic acidmolecule comprises the nucleotide sequence as shown by any of SEQ ID NO:25-30, 38-42 or any combination thereof.

The present invention also provides an expression vector comprising saidnucleic acid molecule as well as a host cell comprising said expressionvector.

The present invention provides methods of producing anti-PD-1 antibodiesor the functional fragments thereof, which comprises: culturing saidhost cells according to the present invention under the condition ofallowing it to produce said antibodies or the functional fragmentsthereof, as well as recovering said antibodies or the functionalfragments thereof produced in this way.

In another aspect, the present invention relates to an immunoconjugatecomprising the antibody or the functional fragment according to thepresent invention that conjugates with a therapeutic agent. Saidtherapeutic agent is preferably toxin, radioisotope, drug or cytotoxicagent.

The present invention also relates to a pharmaceutical compositioncomprising the antibody or the functional fragment thereof according topresent invention as well as a pharmaceutical carrier.

On the other hand, the present invention provides a method used toprevent or treat diseases or conditions through removing, inhibiting orlowering the activity of PD-1, which comprises administration of aneffective treatment dose of the antibody or the functional fragmentthereof according to the present invention, nucleic acid, expressionvector, host cell, immunoconjugate or pharmaceutical composition to asubject in need, wherein: said diseases or conditions are selected fromthe group consisting of cancers, infectious diseases and inflammatorydiseases. Said cancers are preferably selected from the group consistingof melanoma, renal cancer, prostate cancer, breast cancer, colon cancer,lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neckcancer, cutaneous or intraocular malignant melanoma, uterine cancer,ovarian cancer, rectal cancer, anal region cancer, stomach cancer,testicular cancer, uterine cancer, fallopian tube cancer, endometrialcancer, cervical cancer, vaginal cancer, vulva cancer, Hodgkin'sdisease, non-Hodgkin's lymphoma, esophagus cancer, small intestinalcancer, cancer of endocrine system, thyroid cancer, parathyroid cancer,adrenal cancer, soft tissue sarcoma, urethra cancer, penile cancer,chronic or acute leukemia which includes acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, solid tumors during childhood, lymphocytic lymphoma, bladdercancer, kidney or ureter cancer, renal pelvis cancer, vegetation ofcentral nervous system, primary central nervous system lymphoma, tumorangiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid carcinoma, squamous cell cancer, T celllymphoma, environmentally induced cancers which include those induced byasbestos and the combination of said cancers, said infectious diseasesare preferably selected from HIV, influenza, herpes, giardiasis,malaria, leishmaniasis, pathogenic infections caused by the followingviruses: hepatitis viruses (hepatitis A, B and C), herpes virus (such asVZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus,influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus,coronavirus, respiratory syncytial virus, mumps virus, rotavirus,measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus,dengue virus, papilloma virus, molluscum virus, polio virus, rabiesvirus, JC virus and arboviral encephalitis virus, pathogenic infectionsby the following bacteria: chlamydia, rickettsia, mycobacteria,staphylococcus, streptococcus, pneumococcus, meningococcus andconococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, tuberculosis, cholera, tetanus, botulism,anthrax, plague, leptospirosis and Lyme disease bacteria, pathogenicinfections by the following fungi: Candida (Candida albicans, Candidakrusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcusneoformans, aspergillus (Fumigatus, Aspergillus niger, etc.), genus ofMucor (mucor, absidia, rhizopus), Sporothrix schenckii, dermatitis yeastbud, paracoccidiodes brasiliensis, Coccidioides immitis and Histoplasmacapsulatum, pathogenic infections by the following parasites: Entamoebahistolytica, Colon balantidium, fernando's worms, amoeba spine, suctionblow Giardia, cryptosporidium, Pneumocystis carinii, P. vivax, volesBabesia, Trypanosoma brucei, Cruz trypanosoma, Leishmania donovani,Toxoplasma gondii and Nippostrongylus brasiliensis, said inflammatorydiseases are preferably selected from acute disseminatedencephalomyelitis, Addison's disease, ankylosing spondylitis,antiphospholipid antibody syndrome, autoimmune hemolytic anemia,autoimmune hepatitis, arthritis, Behcet's disease, bullous blisteringday sores, celiac disease, Chagas disease, Crohn's disease,dermatomyositis, type-1 diabetes, pulmonary hemorrhage—nephriticsyndrome, graft versus host disease, Graves' disease, Guillain-Banesyndrome, Hashimoto's disease, hyperimmunoglobulin E syndrome,idiopathic thrombocytopenic purpura, lupus erythematosus, multiplesclerosis, myasthenia gravis, pemphigus, pernicious anemia,polymyositis, primary biliary cirrhosis, psoriasis disease, rheumatoidarthritis, Sjogren's syndrome, temporal arteritis, vasculitis, andWegener's granulomatosis.

The present invention also provides the uses of the antibody or thefunctional fragment thereof according to the present invention, nucleicacid, expression vector, host cell, immunoconjugate or pharmaceuticalcomposition in preparing the drugs for treating the diseases orconditions.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the protein size of the extracellular structural region ofPD-1 derived from human (hu) and Macaca fascicularis (cyno) as indicatedby SDS-PAGE.

FIG. 2 shows the combination of biotin-labeled rh-PD-L1 and human Tcells measured by flow cytometry.

FIG. 3 shows that the PD-L1 control, Clone 1, 10, 11, 55 and 64 blockthe binding between the PD-1 on cell surface with the ligand PD-L1 asmeasured by flow cytometry.

FIG. 4 shows the binding of the antibody control, Clone 1, 10, 11, 55and 64 with the PD-1 as well as other CD28 family members (ICOS, CTLA-4and CD28).

FIG. 5 shows the results of the tetanus toxin stimulation experimentsmeasuring the in vitro stimulation of chimeric antibody on T cells.

FIG. 6 shows the secretion levels of IL2 by Clones hu38 (“38”), hu39(“39”), hu41 (“41”) and hu48 (“48”) (control (conIgG4)) of the humanizedantibody measured by the CD8+ cytokine assay kit.

FIG. 7 shows the values of the GFP fluorescence of Clones 38, 39, and 41relative to Clone 48 (control IgG4) of the humanized anti-PD-1 antibodyin experiments where dendritic cells and the modified MD-MAB-453 cellswere cultured together for 3 days followed by the addition of extractedT cells and each of the Clones to culture together for an additional 3days.

FIGS. 8A-8C show the binding between the humanized antibody according tothe present invention with the PD-1 proteins derived from human (FIG.8A), Macaca fascicularis (FIG. 8A), and mouse (FIG. 8C).

FIG. 9 shows the comparison between the PD-1 sequences of human (SEQ IDNO.43), Macaca fascicularis (Cyno) (SEQ ID NO.44) and mouse (SEQ ID NO.45), wherein the major differences between the mouse PD-1 protein andthe human/Macaca fascicularis PD-1 protein are boxed.

FIG. 10 shows the experiment result of T cell proliferation in vitrostimulated by humanized antibody—tetanus antigen memory response.

FIG. 11 shows the experiment result of T cell proliferation in vitrostimulated by humanized antibody—viral polypeptides antigen memoryresponse.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all the technical terms used in this patenthave the same meanings understandable by the ordinary technicians in theart. As for the definitions and terms in the art, the professionals canspecifically refer to Current Protocols in Molecular Biology (Ausubel).The abbreviation of amino acid residues employs a form of the standardcode of 3 letters and/or 1 letter used in the field for each of the 20commonly-used L-amino acids.

The present invention provides an anti-PD-1 antibody and a functionalfragment thereof, which can bind to the programmed cell death 1 (PD-1).The antibody and the functional fragment thereof according to thepresent invention has at least one of the following features: theability to block the interaction between PD-1 and PD-L1 via highaffinity, or bind to PD-1 with high specificity but not to other CD28family members (such as ICOS, CTLA-4 and CD28), or activatetumor-specific T cells to kill the tumor cells and promote CD8+ to enterthe tissue of solid tumor so as to increase greatly the levels of theimmune effectors such as IFNγ.

The present invention also provides a humanized anti-PD-1 antibody and afunctional fragment thereof. Said humanized antibody is obtained bycomputer simulation design of the mouse-derived antibody produced byimmunized mouse in combination with bacteriophage display technology.Its binding epitopes are also identified accordingly based on itsbinding characteristics with PD-1 proteins of various species. Exceptthe advantageous characteristics of the anti-PD-1 antibody and thefunctional fragment thereof described above, said humanized anti-PD-1antibody and the functional fragment thereof according to the presentinvention also binds to PD-1 proteins of human or Macaca fascicularisvia high affinity, but does not interact with the mouse-derived PD-1protein.

On the premise of not substantially influencing the activity of theantibody, those skilled in the art can replace, add and/or remove one ormore (such as 1, 2, 3, 4, 5, 6, 7, 9 or 10 or more) amino acids of thesequence according to the present invention so as to produce a variantof the sequence of said antibody or the functional fragment thereof.They are all deemed to be included in the protection scope of thepresent invention. For example, the amino acid in the variable regioncan be replaced with that of similar property. The sequence of saidvariant according to the present invention can have an identity of atleast 95%, 96%, 97%, 98% or 99% to its source sequence. Said sequenceidentity described in the present invention can be measured by sequenceanalysis software, for example, the computer program BLAST using defaultparameter, especially BLASTP or TBLASTN.

The antibody according to the present invention can be full length (forexample, IgG1 or IgG4 antibody) or comprises only the part that binds anantigen (for example, Fab, F(ab′)2 or scFv fragment), or an antibodythat has been modified to affect its function. The present inventioncomprises the anti-PD-1 antibody comprising modified glycosylationpattern. In some applications, it is useful to conduct modification toremove the undesirable glycosylation sites or avoid the part of fucoseon the oligosaccharide chain to, for example, enhance the antibody ofantibody-dependent cellular cytotoxicity (ADCC) function. In some otherapplications, the modification of galactosylation can be conducted tochange the complement-dependent cytotoxcity (CDC).

The terms used in this patent—“functional fragment” refers to especiallythe antibody fragment such as Fv, scFv (sc refers to single strand),Fab, F(ab′)2, Fab′, scFv-Fc fragment or diabody, or any fragment whosehalf life should be possibly increased by means of chemical modificationor incorporating into the liposome. Said chemical modification is, forexample, adding polyalkylene glycol such as polyethylene glycol(“pegylation, PEGylated”) (referred to as a pegylated fragment such asFv-PEG, scFv-PEG, Fab-PEG, F(ab′)2-PEG or Fab′-PEG) (“PEG” ispolyalkylene glycol) and said fragment has EGFR binding activity.Preferably, said functional fragment comprises a partial sequence of theheavy or light variable chain of their source antibody. Said partialsequence maintains sufficient antigen-binding specificity and affinity,which are the same as those of its source antibody. As for PD-1, theaffinity is preferably at least 1/100, and more preferably at least1/10, of the affinity of its source antibody. Said functional fragmentcomprises at least 5 amino acids and preferably comprises 10, 15, 25, 50and 100 continuous amino acids of its source antibody sequence.

Technicians skilled in the art can clone the DNA molecule encoding saidanti-PD-1 antibody according to the present invention into a vector andthen transform to host cell. In this way, the present invention can alsoprovide a type of recombinant DNA vector, which comprises a DNA moleculeencoding said anti-PD-1 antibody according to the present invention.

Preferably, said recombinant DNA vector is a type of expression vector.Technicians skilled in the art can clone the DNA molecule of saidantibody into the expression vector and transform it into host cell toget antibody by means of induction expression. The expression vectoraccording to the present invention comprises the encoding DNA sequenceof the variable region of the heavy chain, the variable region of thelight chain and/or constant region of the anti-PD-1 antibody.Nevertheless, two types of expression vectors can also be constructedseparately: one comprising the variable region of the heavy chain andconstant region and another comprising the variable region of the lightchain and constant region. The two types of expression vectors are thenintroduced into the same mammal. In one preferred embodiment, saidexpression vector further comprises a promoter and a DNA sequenceencoding the secreting signal peptide as well as at least one type ofdrug-resistant gene used to screen.

The host cells according to the present invention can be prokaryotichost cell, eukaryotic host cell or bacteriophage. Said prokaryotic hostcell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteusmirabilis, etc. Said eukaryotic host cell can be fungi such as Pichiapastoris, Saccharomyces cerevisiae, fission yeast and Trichoderma,insect cells such as Spodoptera frugiperda, plant cells such as tobacco,mammalian cells such as BHK cell, CHO cell, COS cell and myeloma cell.In some embodiments, the host cells according to the present inventionare preferably mammalian cells, more preferably, BHK cells, CHO cells,NSO cells or COS cells.

The term “pharmaceutical composition” used herein refers to thecombination of at least one kind of drug and randomly selectedpharmaceutical carriers or excipients for a special purpose. In someembodiments, said pharmaceutical composition includes the combinations,which are separated in time and/or space provided that they can functionsynergistically to realize the purpose of the present invention. Forexample, the ingredients contained in said pharmaceutical composition(for example, the antibody, nucleic acid molecule, combination and/orconjugate of nucleic acid molecule) can be administered to the subjectas a whole or separately. In the case that the ingredients contained insaid pharmaceutical composition are administered separately to thesubject, they can be used simultaneously or in turn. Preferably, saidpharmaceutical carrier is water, buffer aqueous solution, isotonicsaline solutions such as PBS (phosphate buffer), glucose, mannitol,dextrose, lactose, starch, magnesium stearate, cellulose, magnesiumcarbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkyleneglycols such as polypropylene glycol, triglyceride and others. The typeof the pharmaceutical carrier can be selected based on whether thecomposition according to the present invention is formulated to beadministered via oral, intranasal, intradermal, subcutaneous,intramuscular or intravenous route. The composition according to thepresent invention can comprise a wetting agent, an emulsifier or abuffer solution as additive.

The pharmaceutical composition according to the present invention can beadministered via any appropriate route, for example, oral, intranasal,intradermal, subcutaneous, intramuscular or intravenous.

In one relevant aspect, the present invention provides a pharmaceuticalcomposition comprising an anti-PD-1 antibody and a second therapeuticagent. In one embodiment, the second therapeutic agent is any agentadvantageous to combine with anti-PD-1 antibody. Examples of such agentadvantageous to combine with anti-PD-1 antibody include, but are notlimited to, other agents that can inhibit the activity of PD-1(including the fragments, the peptide inhibitor, the small moleculeantagonists, etc., binding to other antibodies or antigens) and/or theagents that can interfere with the transduction of upstream ordownstream signals of PD-1.

The terms “prevent or treat diseases or conditions through removing,inhibiting or lowering the activity of PD-1” refers to the diseases orconditions caused by expression of PD-1 or those diseases or conditionswith the symptoms/features of PD-1 expression. In some embodiments, saiddiseases or conditions are cancers or infectious diseases. Said cancersinclude, but are not limited to, lung cancer, liver cancer, ovariancancer, cervical cancer, skin cancer, bladder cancer, colon cancer,breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer,oral squamous cell carcinoma, head/neck cancer. Said infectious diseasesinclude, but are not limited to, HIV infection and Hepatitis B virusinfection.

The term “effective treatment dose” used in this patent refers to a dosesufficient to confer benefit to the application target. The doseadministered, the administration rate and the duration may depend on theconditions and severity of the target to treat. The prescription oftreatment (for example, determination of dose) is determined by aphysician, who may consider the factors such as the disease to treat,the condition of individual patient, the administration site, and theadministration method.

The term “subject” used in this patent refers to mammals such as humanor other animals, for example, wild animals (heron, stork, crane, etc),livestock (duck, goose, etc) or laboratory animals (orangutan, monkey,rate, mouse, rabbit and guinea pig, etc)

The following Examples are provided to prove and further explain somepreferred embodiments according to the present invention. Nevertheless,they should not be interpreted as limiting the scope of the presentinvention.

EXAMPLES Example 1. Clone Human PD1 Extracellular Structural Region intoEukaryotic Expression Plasmid

Total RNA from human peripheral blood cells (Beijing Red Cross BloodCenter) was extracted with TRIzol™ RNA extraction kit (Invitrogen) andcDNA was obtained with reverse transcription kit by Invitrogen. The PD1extracellular fragment was obtained by PCR amplification of the cDNAusing the upstream primer 5′-GTACGCTAGCCACCATGCAGATCCCACAGGC-′3 (SEQ IDNO.31) and the downstream primer 5′-GATCCTCGAGCCACCAGGGTTTGGAACTG-′3(SEQ ID NO.32). The amplified product was digested by Nhe I and Xho Iand cloned into the eukaryotic expression plasmid system of pCDNA3.1.293T cells (ATCC) were transfected with this plasmid for 3 days, thesupernatant of the cell culture was collected to purify h-PD1. Total RNAfrom the peripheral blood cells of Macaca fascicularis were alsoextracted and the produced cDNA was cloned into the a eukaryoticexpression vector.

As shown in FIG. 1, due to the posttranslational modification such asglycosylation, the protein sizes of PD-1 extracellular structuralregions of human-derived (hu) and Macaca fascicularis derived (cyno)PD-1 were about 50K Dalton after Coomassie blue staining.

Example 2. Test the Combination of the PD-1 on Cells and the LigandPD-L1

1. Isolate T Cells from Human Peripheral Blood Cells

When the suspension of peripheral blood cells (Beijing Blood Institute)flows through nylon-fiber column (Beijing Hede Biotechnology Company),the B cells, plasma cells, mononuclear cells and some ancillary cellswill adhere selectively to the nylon fiber although most T cells passthrough nylon-fiber column and hence yield the enriched T cellpopulation. The procedure is simply described as follows: take a 50-mlglass syringe, pull out the syringe core and fit a rubber hose with clipinto the syringe nozzle. Tie up some nylon fibers and insert them intothe syringe. Fix the syringe on the support and pour RPMI cell culturefluid at 37° C. to pre-treat the nylon fibers. Close the valve, open thevalve after 0.5 hours to release the cell culture fluid. Dilute the cellfluid to be separated with pre-heated RPMI culture fluid to anappropriate concentration about 5.00×10⁷ cells/ml. Pour the cell fluidinto the syringe and submerge the nylon-fiber column. Cover the syringeand incubate at 37° C. for 1 hour. Open the lower opening, release thefluid slowly (1 drip/min) and collect in a centrifuge tube. Centrifugeat 1000×g for 10 minutes to yield the desired T lymphocytes. 2.Conjugate rh-PD-L1 recombinant protein with biotin

Mix 100 ug rh-PD-L1 recombinant protein (purchased from Beijing SinoBiological Inc.) with the biotin-amino-caproic acid-NHS (Thermo)dissolved in DMSO at a molar ratio of 1:4 and keep the mixture still atroom temperature for 1 hour. Then pass the reaction mixture through G25gel column (Thermo) to separate the biotin-marked rh-PD-L1 and the freebiotins. 3. Combine the biotin-marked rh-PD-L1 with human T cells

rh-PD-L1 recombinant protein conjugated with different concentrations ofbiotins was mixed with 10⁵ T cells isolated according to the methodabove. The mixture was incubated at 4° C. for 15 minutes. After washingthree times with PBS, a streptavidin-allophycocyanin (Thermo) (SA-APC)to 0.2 ug/ml was added and incubated with the mixture at 4° C. for 20minutes. After washing three more times with PBS, samples were measuredat 660 nm using the Beckman Dickson FACSCalibur. As shown in FIG. 2, theresult shows that the PD-L1 protein conjugated with biotin can bind to Tcells.

Example 3. Preparation of Anti Rh-PD-1 Antibody

1. Animal Immunization

10 ug rh-PD-1 recombinant protein of 1 mg/ml as antigen was mixed withequivalent immune adjuvants (Freund adjuvant (Sigma-Aldrich)) andimmunized subcutaneously three female FVB mice of 6-week old. After thefirst immunization, the same dose is administered once per week to boostthe immunization.

2. Cell Fusion

After the last shot of enhanced immunization, lymph nodes at the thighroot of mice were collected and milled in the normal saline. Theproduced suspension, enriched with B cells, was fused with the SP2/0cells via the conventional method of electrophoretic transfer (see BTXelectroporator manual). The fusion cells were cultured at the conditionof 5% CO₂ and 37° C. in the RPMI-1640 whole culture medium containingHAT (Sigma) to culture.

Example 4. Experiment of Blocking Ligand and Receptor

From 20000 of monoclonal hybridoma cell lines, the enzyme label (Elisa)reaction was employed to screen out the 1220 clones of secretedantibodies, which can bind to PD-1 proteins. Five of these 1220 clonesof antibodies had the ability to inhibit the binding between thebiotin-marked PD-L1 and the PD-1 receptor on T cells to various degrees.

1 ug/ml of each of the five antibodies described as above were incubatedwith 312 ng/ml of biotin-marked rh-PD-L1 (concentration) at roomtemperature for 20 minutes. The mixture was then incubated with T cellsisolated from human peripheral blood cells at 4° C. for 15 minutes.After washing three times with normal saline and 0.2 ug/ml of SA-APC wasadded to the mixture and incubated at 4° C. for 15 minutes. Afterwashing three times with normal saline, samples were measured with BD'sflow cytometer to verify whether the antibodies can inhibit the bindingof rh-PD-L1 and PD-1 receptor on the surface of T cell.

As shown in FIG. 3, Clones 1, 10 and 11 can block the binding of PD-1with the ligand PD-L1 on the cell surface, whereas Clones 55 and 64 canonly play a weak inhibition role.

Example 5. Binding of the Antibody Candidates to Other CD28 FamilyMembers

In order to further test the binding specificity of antibody candidateClones 1, 10, 11, 55 and 64, 1 ug/ml of rh-PD-1 or the other CD28 familymembers, i.e., ICOS, CTLA-4 and CD28 (R&D System) in the carbonatebuffer solution (0.05M PH9) was used to coat a 96-well enzyme-labelplate and kept at 4° C. overnight. The next day, solution was removedfrom the wells and the wells were washed three times with a washingbuffer (PBS+0.5% TWEEN). PBS solution containing 3% BSA was added to thewells to block for 20 minutes. After washing three times with thewashing buffer, 100 ul of each antibody clone was added at 1 ug/ml andincubated at room temperature for 1 hour. After three washes with thewashing buffer, HRP cross-linked goat anti mouse antibody (JacksonImmunoresearch) was diluted in the washing buffer 1:0000, and added tothe sample wells and incubated at room temperature for 1 hour. Afterthree washes with the washing buffer, 50 ul TMB (tetramethyl benzidine)substrate solution was added to develop the color. The color reactionwas terminated after the reaction was continued for 10 minutes at roomtemperature by adding 25 ul of 0.5M sulfuric acid solution to thereaction mixture. The absorbance was then measured at 450 nm.

As shown in FIG. 4, all the antibody candidate clones tested canrecognize and bind to rh-PD-1 but not other CD28 family members.

Example 6. The Variable Region Sequence of the Antibody Candidates

Candidate hybridoma cells were cultured until reaching a total count of10′. The cells were collected by centrifugation at 1000 rpm for 10minutes and total RNA from the cells was extracted with TRIzol™ reagentkit (Invitrogen). The total RNA was used as the template to synthesizethe first strand cDNA (Qiagen), which, in turn, was used as a templateto amplify the DNA sequence of the variable region of the respectivehybridoma cells. The primer sequence used in the amplification reactionis complementary to the first framework region and the constant regionof the variable region of antibody (Larrick, J. W., et al., (1990)Scand. J. Immunol., 32, 121-128 and Coloma, J. J. et al., (1991)BioTechniques, 11,152-156 et al.). In a 50 μl reaction system, 1 μl ofcDNA, 5 μl of 10×PCR buffer solution, each 1 μl (25 pmol) of upstreamand downstream primer, 1 μl of dNTP, 1 μl of 25 mmol/L MgCl₂, 39 μl ofH₂O, were added separately. The initial denaturation of the template wasconducted at 95° C. for 10 minutes. 1 μl of Taq enzyme (Invitrogen) wasadded to the reaction to start temperature cycles of the PCRamplification. The reaction conditions are as follows: denaturing at 94°C. for 1 minute, annealing at 58° C. for 1 minute, and extending at 72°C. for 115 minutes. The cycle was repeated 30 times before the reactionmixture was kept at 72° C. for 10 minutes.

The amplified product is sequenced and the sequences of variable regionsof the heavy chain and light chain of Clone 1, 10 and 11 of hybridomaare shown below: Clone 1:

Heavy chain (SEQ ID NO. 19)←------------FR1-------------→ CDR1←----FR2----→QGQLQQSGAELVRPGASVTLTCKASGYTFT DYEMH WVKQTPIHGLEWIG       CDR2      ←-----------FR3---------------→ VIESETGGTAYNQKFKGKAKLTADKSSSTAYMELRSLTSEDSAVYYCTR     CDR3        ←---FR4---→EGITTVATTYYWYFDV WGTGTTVTVSS Nucleic acid sequence (SEQ ID NO. 25)CAGGGTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGACCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTATACATGGCCTGGAATGGATTGGAGTTATTGAATCTGAAACTGGTGGTACTGCCTACAATCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGAGGGTATTACTACGGTAGCAACTACGTACTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTC ALight chain  (SEQ ID NO. 20)←---------FR1---------→      CDR1    ←-----FR2-- DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLE WYLQKPGQSPE ---→ CDR2 ←-------------FR3--------------→LLTY KVYNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC    CDR2  ←---FR4--→FQGSHVPFT FGSGTKLEIK Nucleic acid sequence (SEQ ID NO. 26)GATGTTTTGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAGAGCTCCTGATCTACAAAGTTTACAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAClone 10: Heavy chain(SEQ ID NO. 21) ←------------FR1------------→CDR1←---FR2-----→QVQLQQSGAELVRPGASVTLSCKASGYTFT

WVKQTPVHGLEWIG          CDR2     ←--------FR3------------------→

KAILTADKSSSTAYMELRSLTSEDSAVYYCTR CDR3            ←---FR4---→EGITTSVVTYYWYFDV WGTGTTVTVSS Nucleic acid sequence (SEQ ID NO. 27)CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTGCTGCCTACAATCAGAAGTTCAAGGGCAAGGCCATACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGAGGGTATTACTACGTCAGTGGTTACGTACTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA Light chain (SEQ ID NO. 22)←----------FR1--------→      CDR1     ←----FR2-- DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLE WYLQKPGQSPK ---→ CDR2 ←-------------FR3--------------→LLIY KVSNRFS GVPDRFSGSGSGTDFTLRISRVEPEDLGVYYC  CDR2    ←---FR4--→FQGSHVPLT FGSGTKLEIK Nucleic acid sequence (SEQ ID NO. 28)GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAGGATCAGCAGAGTGGAGCCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCACTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAClone 11: Heavy chain(SEQ ID NO. 23) ←------------FR1-------------→ CDR1←-----FR2----QVTLKESGPGILQPSQTLSLTCSFSGFSLS TFGMGVG WIRQPSGKGLEWL→       CDR2    ←-------------FR3--------------→ A HIWWDDDKYYNPALKSRLTISKNTSKNQVFLKIANVDTEDTATYYCAR I    CDR2  ←----FR4---→ EERFRWYFDVWGTGTTVTVSS Nucleic acid sequence (SEQ ID NO. 29)CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTTTGGTATGGGTGTCGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTACTATAATCCCGCCCTGAAGAGTCGGCTCACAATCTCCAAGAATACCTCCAAAAACCAGGTATTCCTCAAGATCGCCAATGTGGACACTGAAGATACTGCCACATACTACTGTGCTCGAATAGAGGAGAGGTTCCGCTGGTACTTCGATGTCTGGGGCACAGGGACCACGGT CACCGTCTCCTCALight chain (SEQ ID NO. 24)←---------FR1--------→    CDR1    ←-----FR2----- RAGWTQESALTTSPGETVTLTCRSSTGAITTSNYAN WVQEKPDHLFTGLI→ CDR2 ←-------------FR3--------------→ CDR2  ← G GTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHWV F ---FR4--→ GGGTKLTVLNucleic acid sequence (SEQ ID NO. 30)AGGGCTGGTTGGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTATTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA

Example 7. Construct the Expression Vector of Chimeric Antibody

The Fc fragment of the constant region of the heavy chain and the IAconstant region of the light chain from human blood cells (Beijing BloodInstitute) were cloned into the plasmid pCDNA3.1 (see Walls M A, Hsiao Hand Harris L J (1993), Nucleic Acids Research, Vol. 21, No. 122921-2929) for modification. Said sequence fragments of the heavy chainand light chain described in Example 6 were synthesized by GenscriptCorporation. The heavy chain sequence fragment, after digestion by Xho Iand Age I enzymes, and the light chain sequence fragment, afterdigestion by Sma I and Dra III enzymes, were cloned into the plasmidpCDNA3.1 and sequenced to confirm the sequence of cloned DNA. Theexperimental materials in the description below were all obtainedthrough transfecting the cells with this series of plasmids andpurifying the products generated by the transfected cells.

Example 8. In Vitro Stimulation of T Cells by Chimeric Antibody—TetanusToxin Stimulation Test

Freshly prepared peripheral blood mononuclear cell (PBMC) were placedinto 96-well flat-bottom plate. After an overnight incubation, variousconcentrations of antibodies and 100 ng/ml of tetanus toxin (TT) (ListBiological Laboratories) were added. Supernatant was collected from eachof the sample well three days later. The content of IFNγ in thesupernatant was measured by ELISA using the INFNy kit (R&D System). Asshown in FIG. 5, after the PD-1 signal is blocked, the cytokinesecretion by the immune cells activated by the TT stimulation increasedsignificantly. In addition, the titers of the candidate antibodies arehigher than EH12.2H7, an antibody described in the Chinese patentapplication CN 200980147059.0 and purchased from Biolegend Corporation.

Example 9. Interaction of Various Antibodies with PD-1 after Exchange ofHeavy and Light Chains of the Former

Using the method described in Example 7, the light and heavy chains ofthe antibodies 1 and 10 were recombined reciprocally to generaterecombinant antibodies H1L10 (heavy chain 1, light chain 10) and H10L1(heavy chain 10, light chain 1). These antibodies, the original antibody1 (heavy chain 1, light chain 1) and the original antibody 10 (heavychain 10, light chain 10) were tested by ELISAs and the results of EC50sfor these antibodies are shown in the table below:

Sample H1L1 H10L10 H1L10 H10L1 EC50 (pM) 338.1 426.3 270.1 528.1

The result shows that the antibodies produced after recombination canstill bind to PD-1 protein effectively.

Example 10. Humanization Modification of Antibody

The humanization modification was conducted based on the sequence ofvariable region of the antibody secreted by the hybridoma cell obtainedas described above. In brief, the process of humanization modificationinvolved the following steps: A—comparing the gene sequences of theantibodies secreted by various hybridoma cells with the gene sequence ofthe human embryonic antibody to find the sequence of high homology;B—analyzing and testing the affinity of HLA-DR in order to select theframework sequence of human embryo with low affinity; C—using computeranalog technology to apply molecular docking to analyze the sequences offramework amino acids in the variable region and the surrounding, andexamining its spatial stereo binding mode; calculating the electrostaticforce, Van der Waals force, hydrophobicity-hydrophilicity and theentropy value to analyze the key individual amino acids in the genesequences of the antibodies secreted by various hybridoma cell, whichare critical for interacting with PD-1 and maintaining the antibodies'spatial configuration, grafting these key amino acids back to theselected gene framework of human embryo. The amino acid sites in theframework region which must be reserved were also identified and randomprimers were synthesized to construct the phage library. The humanizedantibody library was then screened (Pini, A. et al., (1998). Design andUse of a Phage Display Library: HUMAN ANTIBODIES WITH SUBNANOMOLARAFFINITY AGAINST A MARKER OF ANGIOGENESIS ELUTED FROM A TWO-DIMENSIONALGEL., Journal of Biological Chemistry, 273(34): 21769-21776). A numberof humanized antibodies were obtained from the screening, including thefollowing clones. The light chain sequences of Clone 38, 39 and 41 arethe same, and the heavy chain sequences of Clone 39 and 48 are the same.

Heavy chain of Clone 38 (SEQ ID NO. 33 (peptide); SEQ ID NO. 38(nucleotide)) <-------------FR1------------> CDR1<-----FR2---->QGQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPIHGLEWIG       CDR2      <--------------FR3-------------> VIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR       CDR3      <---FR4--->EGITTVATTYYWYFDV WGQGTTVTVSSCAGGGCCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACGAGATGCACTGGGTGAGACAGGCCCCCATCCACGGCCTGGAGTGGATCGGCGTGATCGAGAGCGAGACCGGCGGCACCGCCTACAACCAGAAGTTCAAGGGCAGAGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCCAGAGAGGGCATCACCACCGTGGCCACCACCTACTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Light chain of Clone 38 (SEQ ID NO. 34 (peptide); SEQ ID NO. 30 (nucleotide))<----------FR1-------->       CDR1     <-----FR2--DVVMTQSPLSLPVTLGQPASISC RSSQSIVHSNGNTYLE WYLQKPGQSPQ---> CDR2  <--------------FR3-------------> LLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQG CDR

----FR4---> SHVPLT FGQGTKLEIKGATGTGGTGATGACCCAGAGCCCGCTGAGCCTGCCGGTGACCCTGGGCCAGCCGGCGAGCATTAGCTGCCGCAGCAGCCAGAGCATTGTGCATAGCAACGGCAACACCTATCTGGAATGGTATCTGCAGAAACCGGGCCAGAGCCCGCAGCTGCTGATTTATAAAGTGAGCAACCGCTTTAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATGTGGGCGTGTATTATTGCTTTCAGGGCAGCCATGTGCCGCTGACCTTTGGCCAGGGCACCAAACTGGAAATTAAA Heavy chain of Clone 39(SEQ ID NO. 35 (peptide); SEQ ID NO. 40 (nucleotide))<-------------FR1------------> CDR1<-----FR2---->QGQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG       CDR2      <--------------FR3------------> VIESETGGTAYNQKFKGRAKITADKSTSTAYMELSSLRSEDTAVYYCT        CDR3      <---FR4--->REGITTVATTYYWYFDV WGQGTTVTVSSCAGGGCCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACGAGATGCACTGGGTGAGACAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCGTGATCGAGAGCGAGACCGGCGGCACCGCCTACAACCAGAAGTTCAAGGGCAGAGCCAAGATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCACCAGAGAGGGCATCACCACCGTGGCCACCACCTACTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Light chain of Clone 39(SEQ ID NO. 34 (peptide); SEQ ID NO. 30 (nucleotide))<----------FR1-------->       CDR1     <-----FR2--DVVMTQSPLSLPVTLGQPASISC RSSQSIVHSNGNTYLE WYLQKPGQSPQ---> CDR2  <--------------FR3-------------> LLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC    CDR3  <---FR4--> FQGSHVPLTFGQGTKLEIK GATGTGGTGATGACCCAGAGCCCGCTGAGCCTGCCGGTGACCCTGGGCCAGCCGGCGAGCATTAGCTGCCGCAGCAGCCAGAGCATTGTGCATAGCAACGGCAACACCTATCTGGAATGGTATCTGCAGAAACCGGGCCAGAGCCCGCAGCTGCTGATTTATAAAGTGAGCAACCGCTTTAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATGTGGGCGTGTATTATTGCTTTCAGGGCAGCCATGTGCCGCTGACCTTTGGCCAGGGCACCAAACTGGAAATTAAA Heavy chain of Clone 41(SEQ ID NO. 36 (peptide); SEQ ID NO. 41 (nucleotide))<-------------FR1------------> CDR1<-----FR2---->QGQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG       CDR2      <--------------FR3------------> VIESETGGTAYNQKFQGRVTLTADKSSSTAYMELSSLRSEDTAVYYCT        CDR3      <----FR4-->REGITTVATTYYWYFDV WGQGTLVTVSSCAGGGCCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCTATACCTTTACCGATTATGAAATGCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCGTGATTGAAAGCGAAACCGGCGGCACCGCGTATAACCAGAAATTTCAGGGCCGCGTGACCCTGACCGCGGATAAAAGCAGCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCACCCGCGAAGGCATTACCACCGTGGCGACCACCTATTATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC Light chain of Clone 41(SEQ ID NO. 34 (peptide); SEQ ID NO. 30 (nucleotide))<----------FR1-------->       CDR1     <-----FR2--DVVMTQSPLSLPVTLGQPASISC RSSQSIVHSNGNTYLE WYLQKPGQSPQ---> CDR2  <--------------FR3-------------> LLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC    CDR3  <--FR4---> FQGSHVPLTFGQGTKLEIK GATGTGGTGATGACCCAGAGCCCGCTGAGCCTGCCGGTGACCCTGGGCCAGCCGGCGAGCATTAGCTGCCGCAGCAGCCAGAGCATTGTGCATAGCAACGGCAACACCTATCTGGAATGGTATCTGCAGAAACCGGGCCAGAGCCCGCAGCTGCTGATTTATAAAGTGAGCAACCGCTTTAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATGTGGGCGTGTATTATTGCTTTCAGGGCAGCCATGTGCCGCTGACCTTTGGCCAGGGCACCAAACTGGAAATTAAA Heavy chain of Clone 48(SEQ ID NO. 35 (peptide); SEQ ID NO. 40 (nucleotide))<-------------FR1------------> CDR1<-----FR2---->QGQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG       CDR2      <--------------FR3------------> VIESETGGTAYNQKFKGRAKITADKSTSTAYMELSSLRSEDTAVYYCT        CDR3      <---FR4--->REGITTVATTYYWYFDV WGQGTTVTVSSCAGGGCCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACGAGATGCACTGGGTGAGACAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCGTGATCGAGAGCGAGACCGGCGGCACCGCCTACAACCAGAAGTTCAAGGGCAGAGCCAAGATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCACCAGAGAGGGCATCACCACCGTGGCCACCACCTACTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Light chain of Clone 48(SEQ ID NO. 37 (peptide); SEQ ID NO. 42 (nucleotide))<----------FR1-------->       CDR1     <-----FR2--DVVMTQSPLSLPVTLGQPASISC RSSQSIVHSNGNTYLE WYLQKPGQSPR---> CDR2  <--------------FR3-------------> LLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC    CDR

----FR4---> FQGSHVPLT FGQGTKLEIKGATGTGGTGATGACCCAGAGCCCGCTGAGCCTGCCGGTGACCCTGGGCCAGCCGGCGAGCATTAGCTGCCGCAGCAGCCAGAGCATTGTGCATAGCAACGGCAACACCTATCTGGAATGGTATCTGCAGAAACCGGGCCAGAGCCCGCGCCTGCTGATTTATAAAGTGAGCAACCGCTTTAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATGTGGGCGTGTATTATTGCTTTCAGGGCAGCCATGTGCCGCTGACCTTTGGCCAGGGCACCAAACTGGAAATTAAA

Example 11. In Vitro Stimulation of T Cells by Humanized Antibody

Freshly prepared PBMCs (Beijing Blood Institute) were placed in thewells of a 96-well flat-bottom plate. After an overnight incubation, 10ug/ml of antibody and 100 ng/ml of tetanus toxin (TT) were added to thePBMCs. After culturing it for 3 days, the supernatant was collected andthe secretion level of IL2 of Clones 38, 39, 41, 48, and conIgG4(control antibody) of the humanized antibodies were measured usingLuminex® (Thermo Fisher Scientific, Inc.) and CD8+ cytokine assaydetection kit (EMD Millipore, Inc.). The result (see FIG. 6) shows thatall the humanized antibodies can stimulate the T cells.

Example 12. Humanized Antibody can Stimulate T Cells to Kill Tumor CellsIn Vitro

MD-MAB-453 cells were infected with the lentivirus (Qiagen) expressingPD-L1 protein to generate a MD-MAB-453 cell line, which stably expressesPD-L1. A GFP gene was also introduced to said cell line to allow thestable expression of the GFP protein. Dendritic cells (DC) isolated fromthe fresh human peripheral blood cells 300 cells/well were cultured withsaid MD-MAB-453 cells stably expressing both PD-L1 and GFP (300cells/well) in a 96-well plate together for three days. T cells (1000cells/well) isolated from human peripheral blood and 10 ug/ml of thehumanized anti-PD-1 antibody Clones 38, 39, 41, 48 or the controlantibody “conIgG4” were added to the mixture and cultured together for 3days to before the GFP fluorescence was measured. The result (see FIG.7) shows that all the humanized antibodies can stimulate T cells to killthe tumor cells.

Example 13. The Binding of Humanized Antibodies with PD-1 ProteinsDerived from Various Species

1 ug/ml of human-derived PD-1, Macaca fascicularis PD-1, andmouse-derived PD-1 (Sinobiological) in the carbonate buffer solution(0.05 M PH9) were prepared and used to coat the wells of the 96-wellflat-bottom plates at 4° C. overnight. The next day, solution wasremoved from the wells and the wells were washed three times with awashing buffer. PBS solution containing 3% BSA was added to block thewells for 20 minutes. The wells were then washed three times with thewashing buffer before 100 ul of various concentrations of candidateantibodies were added. The mixture was incubated at room temperature for1 hour and then washed three times with a washing buffer. A HRPconjugated goat anti human antibody (Jackson Immunoresearch) was firstdiluted to 1:10000 with the washing buffer and then added to the wellsto incubate at room temperature for 1 hour. After washing three timeswith the washing buffer, 50 ul TMB substrate solution was added to thewells to develop the color. After 10 minutes at room temperature, thecolor development reaction was terminated with 25 ul 0.5M sulfuric acidsolution and the absorbance was read at 450 nm. The results (FIGS. 8A, Band C) show that all clones bind to PD-1 of human or Macaca fasciculariswith similar affinity, but not with the mouse-derived PD-1.

Compare PD-1 sequences of human, Macaca fascicularis and mouse (see FIG.9 in which the main different regions of mouse PD-1 and PD-1 ofhuman/Macaca fascicularis are boxed). Experimental results have provedthat the epitope of the candidate antibody that binds to PD-1 proteinexists in these regions.

Example 14. Experiment of In Vitro Stimulation by Humanized Antibody toT Cell Proliferation—Tetanus Antigen Memory Response Experiment

Freshly prepared PBMC (Beijing Blood Institute) were placed into thewells of a 96-well flat-bottom plate. After incubation overnight, thecells were labeled with carboxyfluorescein succinimidyl ester (CFSE) andincubated with 10 ug/ml of humanized antibody (38, 39, 41 and 48) and100 ng/ml of tetanus toxin (TT) (List Biological Laboratories). T cellproliferation at the 6^(th), 8^(th) and 10^(th) day was analyzed basedon the dilution ratio of CSFE with the flow cytometry (FACS). As shownby the result of FIG. 10, compared with the control IgG the immune cellsactivated by TT stimulation induced through blocking the PD-1 signal wasunder further division and proliferation.

Example 15. In Vitro Stimulation of T Cell Proliferation by HumanizedAntibody—Viral Polypeptides Antigen Memory Response Experiment

Freshly prepared PBMCs (Beijing Blood Institute) were placed into thewells of a 96-well flat-bottom plate. After an overnight incubation, thecells were labeled with CFSE. 10 ug/ml of humanized antibody (38, 39, 41and 48) and 1 ug/ml of peptide mixture of CMV, EBV and Influenza (“CEF”)were added to the wells. Quantitative analysis were conducted on T cellproliferation at the 6th, 8th and 10th day with the flow cytometry(FACS) based on the dilution ratio of CSFE. As shown by the result ofFIG. 11, compared with the control NC; the immune cells activated by CEFmixed polypeptides stimulation induced through blocking the PD-1 signalwas under further division and proliferation.

1-14. (canceled)
 15. A method used to prevent or treat diseases orconditions through removing, inhibiting or lowering the activity ofPD-1, which comprises administration of effective treatment dose of theantibody or functional fragment thereof according to claim 1 or apharmaceutical composition comprising the antibody or functionalfragment thereof to a subject in need, where said diseases or conditionsare selected from the group consisting of cancers, infectious diseasesor inflammatory diseases, said cancers are preferably selected frommelanoma, renal cancer, prostate cancer, breast cancer, colon cancer,lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neckcancer, cutaneous or intraocular malignant melanoma, uterine cancer,ovarian cancer, rectal cancer, anal region cancer, stomach cancer,testicular cancer, uterine cancer, fallopian tube cancer, endometrialcancer, cervical cancer, vaginal cancer, vulva cancer, Hodgkin'sdisease, non-Hodgkin's lymphoma, esophagus cancer, small intestinalcancer, endocrine system cancer, thyroid cancer, parathyroid cancer,adrenal cancer, soft tissue sarcoma, urethra cancer, penile cancer,chronic or acute leukemia which includes acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, solid tumors during childhood, lymphocytic lymphoma, bladdercancer, kidney or ureter cancer, renal pelvis cancer, central nervoussystem vegetation, primary central nervous system lymphoma, tumorangiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid carcinoma, squamous cell cancer, T celllymphoma, environmentally induced cancers which include those induced byasbestos and the combination of said cancers, said infectious diseasesare preferably selected from HIV, influenza, herpes, giardiasis,malaria, leishmaniasis, pathogenic infections caused by the followingviruses: hepatitis viruses (hepatitis A, B and C), herpes virus (such asVZV, HSV-1, HAV-6, HSV-11, and CMV, Epstein Barr virus), adenovirus,influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus,coronavirus, respiratory syncytial virus, mumps virus, rotavirus,measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus,dengue virus, papilloma virus, molluscum virus, polio virus, rabiesvirus, JC virus and arboviral encephalitis virus, pathogenic infectionsby the following bacteria: chlamydia, rickettsia, mycobacteria,staphylococcus, streptococcus, pneumococcus, meningococcus andconococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, tuberculosis, cholera, tetanus, botulism,anthrax, plague, leptospirosis and Lyme disease bacteria, pathogenicinfections by the following fungi: Candida (Candida albicans, Candidakrusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcusneoformans, aspergillus (fumigatus, Aspergillus niger, etc.), genus ofMucor (mucor, absidia, rhizopus), Sporothrix schenckii, dermatitis yeastbud, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasmacapsulatum, pathogenic infections by the following parasites: Entamoebahistolytica, Colon balantidium, fernando's worms, amoeba spine, suctionblow Giardia, cryptosporidium, Pneumocystis carinii, P. vivax, volesBabesia, Trypanosoma brucei, Cruz trypanosoma, Leishmania donovani,Toxoplasma gondii and Nippostrongylus brasiliensis, said inflammatorydiseases are preferably selected from acute disseminatedencephalomyelitis, Addison's disease, ankylosing spondylitis,antiphospholipid antibody syndrome, autoimmune hemolytic anemia,autoimmune hepatitis, arthritis, Behcet's disease, bullous blisteringday sores, celiac disease, Chagas disease, Crohn's disease,dermatomyositis, type-I diabetes, pulmonary hemorrhage—nephriticsyndrome, graft versus host disease, Graves' disease, Guillain-Barresyndrome, Hashimoto's disease, hyperimmunoglobulin E syndrome,idiopathic thrombocytopenic purpura, lupus erythematosus, multiplesclerosis, myasthenia gravis, pemphigus, pernicious anemia,polymyositis, primary biliary cirrhosis, psoriasis disease, rheumatoidarthritis, Sjogren's syndrome, temporal arteritis, vasculitis andWegener's granulomatosis. wherein the antibody or a functional fragmentthereof that can bind to the programmed cell death 1 (PD-1) proteinhaving an amino acid sequence of SEQ ID NO: 43, wherein the antibody ora functional fragment thereof comprises: i) a heavy chain CDR1 havingthe amino acid sequence of Asp Tyr Glu Met His (SEQ ID NO: 1), ii) aheavy chain CDR2 having the amino acid sequence of Val Ile Glu Ser GluThr Gly Thr Ala Tyr Asn Gin Lys Phe Lys (SEQ ID NO: 2), iii) a heavychain CDR3 having the amino acid sequence of Glu Gly Ile Thr Val Ala ThrTyr Trp Tyr Phe Asp Val (SEQ ID NO: 3), iv) a light chain CDR1 havingthe amino acid sequence of Arg Ser Gin Ser Ile Val His Ser Asn Gly AsnThr Tyr Leu Glu (SEQ ID NO: 10), v) a light chain CDR2 having the aminoacid sequence of Lys Val Ser Asn Arg Phe Ser (SEQ ID NO: 11), and vi) alight chain CDR3 having the amino acid sequence of Phe Gin Gly Ser HisVal Pro Leu Thr (SEQ ID NO: 12).
 16. The method of claim 15, wherein theantibody or a functional fragment thereof comprises: (i) a heavy chainvariable region having the amino acid sequence of SEQ ID NO: 19, or anamino acid sequence having at least 95% sequence identity with SEQ IDNO: 19, and/or (ii) a light chain variable region having the amino acidsequence of SEQ ID NO: 22, or an amino acid sequence having at least 95%sequence identity with SEQ ID NO:
 22. 17. The method of claim 15,wherein the antibody or functional fragment thereof is a chimericantibody or a humanized antibody.
 18. The method of claim 15, whereinthe antibody or functional fragment thereof comprises: i) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 35 oran amino acid sequence that has at least 95% sequence identity with SEQID NO: 35, and/or ii) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 37 or an amino acid sequence that has atleast 95% sequence identity with SEQ ID NO:
 37. 19. The method of claim15, wherein the antibody or functional fragment thereof comprises (i) aheavy chain variable region comprising SEQ ID NO: 35 or an amino acidsequence that has at least 95% sequence identity with SEQ ID NO: 35 and(ii) a light chain variable region comprising SEQ ID NO: 37 or an aminoacid sequence that has at least 95% sequence identity with SEQ ID NO:37.
 20. The method of claim 15, wherein the antibody or functionalfragment thereof is coupled with a therapeutic agent, wherein saidtherapeutic agent is a toxin, radioisotope, drug, or cytotoxic agent.